Kaguni J M, Kornberg A
J Biol Chem. 1982 May 25;257(10):5437-43.
RNA polymerase specifically primes the replication of M13 DNA but not phi X174 DNA in vivo and in crude extracts of Escherichia coli. Yet purified preparations of RNA polymerase have been observed not to distinguish between the two templates. We investigated the basis for specificity by assaying priming and transcriptional activities on single-stranded phage DNAs covered by single-stranded DNA-binding protein. In the course of preparing homogeneous RNA polymerase holoenzyme, loss of specificity and decreased priming activity resulted from procedures which removed the rho subunit. Specificity was restored and priming activity was increased upon the addition of rho subunit to core RNA polymerase. Priming of replication depended on a very limited transcription, presumably confined to the unique sequence in M13 DNA that directs the origin of complementary strand synthesis. No transcription was observed on phi X174 DNA comparably covered by binding protein. Thus, the specific priming of M13 DNA replication in E. coli depends on recognition of an origin sequence by an RNA polymerase holoenzyme with a functional rho subunit. Priming specificity for M13 DNA replication thus provides a sensitive and simple test for the activity of the rho subunit of E. coli RNA polymerase, even though the M13 origin region lacks the sequences characteristic of RNA polymerase promoters.
